Quantification of apolipoprotein D by an immunoassay with time-resolved fluorescence spectroscopy

Abstract

Apolipoprotein D (apoD), also known as gross cystic disease fluid protein-24 (GCDFP-24), is a minor protein moiety of high-density lipoproteins in human plasma. ApoD is expressed in a subset of breast carcinomas and has been proposed as a tumor marker and prognostic indicator for breast cancer progression. Here we describe a new sensitive time-resolved fluorimetric immunoassay for quantification of human apoD in biological specimens using affinity-purified polyclonal anti-human apoD rabbit antibodies and Eu 3+ as a specific probe. Both purified apoD and normal human pool-serum served as reliable primary and secondary standards in the direct sandwich dissociation-enhanced lanthanide fluorescence immunoassay (DELFIA). Plasma apoD concentrations measured by the DELFIA were 99.6±32 μg/ml. The detection limit of the DELFIA procedure was 0.5 ng/ml after sample dilution of 1/8000. The intra-assay coefficient of variation averaged 3.5%, whereas the inter-assay coefficient of variation averaged 6.9%. The concentration of apoD in breast cyst fluids ranged from 6.82 to 28.37 mg/ml. Based on the low detection limit and the high specificity of the DELFIA procedure, we have applied this technique for the measurement of apoD in breast cancer cell supernatants. In estrogen-receptor positive cells, i.e., T-47D and ZR-75-1 cells, 42.6±1.4 and 2.7±0.2 ng apoD/ml supernatant after 4 days in culture without induction of apoD synthesis were measured. A comparison of the direct sandwich DELFIA procedure with an electroimmunoassay commonly used to assay apoD revealed correlation coefficients of 0.986 (serum) and 0.975 (cyst fluids). The present findings indicate that the direct sandwich DELFIA is appropriate for apoD quantification in plasma and breast cyst fluids. Furthermore, the technique should permit studies on the induction of apoD synthesis in the low picomolar range in different carcinoma cells to gain insight into the expression of this atypical apolipoprotein.